TWI786142B - Systems and methods for confirming activation of biological indicators - Google Patents

Abstract

Biological indicators may be improperly activated. The disclosed subject matter is directed to methods of confirming that a biological indicator having an ampule containing a growth medium has been properly activated such that it may be assayed. The methods may include the steps of measuring a first fluorescence intensity of the biological indicator, heating the biological indicator; quenching the fluorescence intensity of the biological indicator from the first fluorescence intensity to a second fluorescence intensity, measuring the second fluorescence intensity; comparing the second fluorescence intensity and first fluorescence intensity to obtain a comparison value; and determining that the comparison value corresponds to a quenching metric of the liquid growth medium.

Description

Systems and methods for confirming activation of biological indicators

The subject matter disclosed herein relates to self-contained biological sterilization indicators.

Medical devices are generally sterilized prior to use to minimize the possibility of using a contaminated device on a subject, which could result in infection of the subject. Various sterilization techniques can be employed, such as steam sterilization, hydrogen peroxide sterilization, and vapor phase sterilization with or without gas plasma and ethylene oxide (EtO). Each of these methods depends to some extent on the rate of diffusion of the sterilizing fluid, typically a gas, over the medical device to be sterilized.

Prior to sterilization, medical devices are typically packaged within containers or pouches with semipermeable barriers that allow the transfer of a sterilizing fluid (sometimes called a sterilant) but prevent the entry of contaminating organisms, especially during sterilization Afterwards, and until medical personnel open the package. For a sterilization cycle to be effective, the contaminating organisms within the package must be killed, since any organisms that survive the sterilization cycle may multiply and re-contaminate the medical device.

While packaging helps prevent contamination of sterile medical devices, packaging can make it difficult to achieve a successful sterilization cycle because the packaging prevents the sterilant from reaching the device or instrument contained therein. This is particularly a problem for devices and instruments that have diffusion-confined spaces therein, as these reduce the likelihood that a sterilization cycle will be effective. For example, endoscopes typically have long narrow lumens into which the sterilant must diffuse in sufficient concentration and for sufficient time to achieve a successful sterilization cycle.

Validating that the sterilization cycle is effective helps medical personnel avoid using contaminated medical devices on subjects. In general, the sterilized medical device itself is not inspected for contaminating organisms because such actions may introduce other contaminating organisms into the medical device, thereby recontaminating the medical device. Therefore, indirect checks were developed in the form of sterilization indicators.

A sterilization indicator is a device that can be placed next to or adjacent to a medical device that is subjected to a sterilization cycle such that the sterilization indicator is subjected to the same sterilization cycle as the medical device. For example, a biological indicator having a predetermined number of microorganisms known to be resistant to the sterilant may be placed in the sterilization chamber next to the medical device and subjected to a sterilization cycle. After the cycle is complete, the microorganisms in the biological indicator can be cultured to determine whether any microorganisms survived the cycle.

Some biological indicators are called "self-contained". These biological indicators generally include: a housing that contains a population of microorganisms; and a source of growth medium in a frangible container that is located in the vicinity of the microorganisms. Like other biological indicators, "self-contained" biological indicators ("SCBI") can be subjected to sterilization cycles with medical devices, such as the STERRAD® system at Advanced Sterilization Products (a division of Ethicon US, LLC, a Johnson & Johnson company) , STERRAD® NX system or STERRAD® 100NX system. After cycling, the frangible container may break to release the growth medium and cultivate any surviving microorganisms in situ. SCBI can be cultured at elevated temperatures (typically about 50°C to 60°C) that promote the growth of viable microorganisms. Culturing using commercially available products typically lasts about twenty-four hours. During this time, it is desirable for medical personnel not to use the medical device until the effectiveness of sterilization has been confirmed. This can lead to inefficiencies in inventory management for healthcare providers, such as hospitals, because, for example, medical devices should be stored when they are not available, which may require healthcare providers to keep more medical devices in their inventory than they would otherwise, To ensure sufficient supply of medical devices. Alternatively, the healthcare provider may use the medical device until the culture is complete and sterilization efficacy confirmed. However, use of a medical device before sterilization efficacy has been confirmed may expose the subject of the medical procedure to the risk of infection from the medical device.

After incubation, the SCBI was analyzed to detect the presence of viable microorganisms. If any microorganisms are detected, some SCBIs are designed to incorporate a growth medium that changes color in the presence of microorganisms. If a color change is detected, the sterilization cycle may be considered invalid. If no microorganisms are detected, the sterilization cycle is considered valid. This color change is attributable to a pH shift due to acid production by living microorganisms metabolizing the growth medium, which also contains the pH indicating dye. Other SCBIs are designed to incorporate growth media that include a fluorophore whose fluorescence depends on the amount of viable microorganisms contained in the media. For these SCBIs, a change in color or in the amount of fluorescence indicates that surviving microorganisms may have multiplied during the culture period.

Fragile containers for SCBI containing liquid growth media are often made of glass. The glass must be strong enough to avoid breakage during transport, eg, from SCBI's manufacturer to a healthcare provider. However, this robustness corresponds to the greater force required by medical personnel to break the ampoule at the desired time. Therefore, some SCBI manufacturers provide hospital personnel with activation devices to assist them in breaking ampoules.

The disclosed subject matter is related to confirming that the biological indicator with the ampule containing the growth medium has been properly activated so that it can be tested after the sterilization process to confirm that the sterilization process should have been effective. The method may comprise the steps of: depressing the cap of the biological indicator; breaking the ampoule; positioning the biological indicator into a biological indicator analyzer having a heating element and a fluorescent sensor, activating the heating element, Measuring the first fluorescence intensity of the biological indicator, heating the biological indicator; quenching the fluorescence intensity of the biological indicator from the first fluorescence intensity to a second fluorescence intensity, measuring the second fluorescence intensity; comparing the second fluorescence intensity with the first fluorescence intensity to obtain a comparison value; and determining that the comparison value corresponds to a quenching measure of the liquid growth medium. In some embodiments, the step of quenching the fluorescence intensity of the biological indicator includes heating the growth medium and the housing of the biological indicator. In some embodiments, the step of quenching the fluorescent intensity of the biological indicator comprises heating the growth medium and the housing of the biological indicator from a temperature between about 22 degrees Celsius and 25 degrees Celsius to about 57 degrees Celsius. degrees of temperature. Additionally, the method may also include the step of cooling the biological indicator prior to the step of positioning the biological indicator into the biological indicator analyzer. In embodiments where the biological indicator is cooled, the step of quenching the fluorescence intensity of the biological indicator may further comprise heating the biological indicator. In some embodiments, the comparison value is the difference between the second fluorescence intensity and the first fluorescence intensity. In other embodiments, the comparison value is a ratio of the second fluorescent intensity to the first fluorescent intensity. In some embodiments, the second fluorescence intensity is measured after the first fluorescence intensity. For example, in some embodiments, the second fluorescence intensity is measured about 210 seconds after the first fluorescence intensity, and the first fluorescence intensity is measured after localizing the biological indicator to the biological indicator assay. Measured about 70 seconds after instrumentation. Alternatively, the first fluorescent intensity is measured about 70 seconds after activating the heating element of the biological indicator analyzer. In some embodiments, the method further includes determining that the first fluorescence intensity value is between a minimum threshold value and a maximum threshold value. For example, in some embodiments, the minimum threshold value is about 0.02 μW/cm ² and the maximum threshold value is about 0.10 μW/cm ² .

The method may also include the steps of: depressing the cover of the biological indicator; breaking the ampoule; positioning the biological indicator into a biological indicator analyzer having a heating element and a fluorescent sensor, activating the heating element 1. Measuring the first fluorescence intensity of the biological indicator, heating the biological indicator; quenching the fluorescence intensity of the biological indicator from the first fluorescence intensity to a second fluorescence intensity, measuring the second fluorescence intensity light intensity; comparing the second fluorescence intensity with the first fluorescence intensity to obtain a comparison value; and determining that the comparison value does not correspond to a quenching measure of the liquid growth medium. In some embodiments, the step of quenching the fluorescence intensity of the biological indicator includes heating the growth medium and the housing of the biological indicator. In some embodiments, the step of quenching the fluorescent intensity of the biological indicator comprises heating the growth medium and the housing of the biological indicator from a temperature between about 22 degrees Celsius and 25 degrees Celsius to about 57 degrees Celsius. degrees of temperature. Additionally, the method may also include stopping cooling of the biological indicator prior to the step of positioning the biological indicator into the biological indicator analyzer. In embodiments where the biological indicator is cooled, the step of quenching the fluorescence intensity of the biological indicator may further comprise heating the biological indicator. In some embodiments, the comparison value is the difference between the second fluorescence intensity and the first fluorescence intensity. In other embodiments, the comparison value is a ratio of the second fluorescent intensity to the first fluorescent intensity. In some embodiments, the second fluorescence intensity is measured after the first fluorescence intensity. For example, in some embodiments, the second fluorescence intensity is measured about 210 seconds after the first fluorescence intensity, and the first fluorescence intensity is measured after localizing the biological indicator to the biological indicator assay. Measured about 70 seconds after instrumentation. Alternatively, the first fluorescent intensity is measured about 70 seconds after activating the heating element of the biological indicator analyzer. In some embodiments, the method further includes determining that the first fluorescence intensity value is between a minimum threshold value and a maximum threshold value. For example, in some embodiments, the minimum threshold value is about 0.02 μW/cm ² and the maximum threshold value is about 0.10 μW/cm ² .

Biological indicators may have properties and characteristics suitable for carrying out methods related to the present subject matter. In some embodiments, a biological indicator can include an ampoule and a housing, and a liquid growth medium contained in the ampoule, wherein the liquid growth medium includes a quencher. In some embodiments, the quencher is not oxygen. The quencher can be selected from the group consisting of aniline, bromobenzene, acrylamide, hydrogen peroxide, imidazole, indole, and succinimide. Alternatively, the quencher may be a metal ion, such as a metal ion selected from the group consisting of Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Ag+, Cr3+, and Fe3+. In some embodiments, the quencher can be oxygen included in the growth medium at a concentration greater than about 37 mg/L. In some embodiments, the quencher can be oxygen included in the growth medium at a concentration greater than about 40 mg/L. In some embodiments, the housing of the biological indicator comprises UV transparent material. In some embodiments, the UV transparent material comprises quartz. In other embodiments, the UV transparent material comprises cyclic olefins.

100‧‧‧Self-contained biological indicator/SCBI

102‧‧‧Shell

104‧‧‧Cover

106‧‧‧convex part

108‧‧‧Chemical indicators

109‧‧‧through hole

110‧‧‧carrier

112‧‧‧ampoules

114‧‧‧first end/first dome

116‧‧‧Second End/Second Dome

118‧‧‧side wall

120‧‧‧plug-ins

122‧‧‧platform

124‧‧‧top surface

126‧‧‧bottom surface

127‧‧‧side wall

128‧‧‧support surface/support

130‧‧‧well

142‧‧‧bottom wall

144‧‧‧bottom wall

146‧‧‧compression force

150‧‧‧holes

200‧‧‧Biological Indicator Analyzer/BIA

210‧‧‧well

212‧‧‧Heating element

220‧‧‧processor

230‧‧‧Light source

240‧‧‧sensors

250‧‧‧Displays

280‧‧‧memory/storage device

300‧‧‧method

310‧‧‧step

320‧‧‧step

330‧‧‧step

340‧‧‧step

350‧‧‧step

360‧‧‧steps

370‧‧‧step

380‧‧‧step

390‧‧‧step

400‧‧‧steps

410‧‧‧step

420‧‧‧step

430‧‧‧step

While the specification concludes with particularity and clear claim to claims for the subject matter described herein, it is believed that the subject matter will be better understood from the following description of certain examples taken in conjunction with the accompanying drawings, in which Like reference numerals identify like elements and wherein: FIG. 1 depicts a side view of a cross-section of a biological indicator; FIG. 2 depicts an exploded view of the biological indicator of FIG. 1; FIG. 3 depicts a biological indicator analyzer in block diagram form; and 4 is a flowchart of an exemplary method for confirming the activation of the biological indicators of FIGS. 1 and 2 , which may be performed by the biological indicator analyzer of FIG. 3 .

The following description sets forth some illustrative examples of claimed subject matter. Other examples, features, aspects, embodiments, and advantages of the present technology will become apparent to those having ordinary skill in the art from the following description. Accordingly, the drawings and descriptions should be regarded as illustrative in nature.

Referring to FIGS. 1 and 2 , a self-contained biological indicator (“SCBI”) 100 is shown. SCBI 100 includes a housing 102 and a cover 104 coupled to the housing. The cover 104 includes a protrusion 106 having a flat, angled, arcuate, annular, or conical shape, or some combination thereof. Cover 104 may further include a chemical indicator 108 that changes color upon exposure to, for example, a chemical sterilant such as hydrogen peroxide. Cover 104 may also include one or more through holes 109 to facilitate the passage of gases (eg, air or sterilant) into or out of the SCBI. The cover 104 is coupled to the housing 102 in a first position and is movable from the first position to a second position. In the first position, the cover 104 is coupled to the housing 102 in such a way that gas (eg, air or sterilant) can move from the surrounding environment and into the SCBI (or vice versa). In this position, any through-holes in the cover 104 are provided above the housing 102 to bring the interior of the housing 102 into fluid communication with the surrounding environment, which permits the introduction and extraction of sterilant into and out of the SCBI 100 . The cover 104 is depressively movable relative to the housing 102 into the second position. In this second position, the through-hole 109 is provided below a top end of the housing 102, which results in a tight fit between the housing 102 and the cover 104, and blocks the through-hole, effectively disabling the SCBI. The interior of 100 is sealed from the surrounding environment.

SCBI 100 also includes a source of microorganisms or active enzymes, such as a carrier 110 impregnated with bacterial spores, other forms of bacteria (eg, bacterial vegetatives), and/or active enzymes. Spores from Bacillus, Geobacillus, and Clostridia species are often used to monitor sterilization processes utilizing saturated steam, hydrogen peroxide, dry heat, gamma irradiation, and ethylene oxide. Accordingly, carrier 110 may be impregnated with spores from Bacillus, Bacillus, and/or Clostridium species. The carrier 110 may be a water absorbing agent, and may be formed of filter paper. Sheet-like materials such as cloth, non-woven polypropylene, rayon, or nylon, and microporous polymeric materials may also be used. Non-absorbent materials are also suitable for use, such as metal (eg, aluminum or stainless steel), glass (eg, glass beads or fiberglass), porcelain, or plastic. Additionally, carrier 110 may be constructed from combinations of the aforementioned materials. In some embodiments, the carrier 110 may have a thickness of about 0.1 to 0.5 mm.

The microorganisms or other sources of biological activity on the carrier 110 can be selected based on the resistance of the source to the particular sterilization process to be used in the sterilization cycle. For example, for a steam sterilization process, Geobacillus stearothermophilus or its spores can be used. For the ethylene oxide sterilization process, Bacillus atrophaeus (formerly known as Bacillus subtilis ) or its spores can be used. In some sterilization processes, the spores resistant to the sterilization process may include but are not limited to at least one of the following: Bacillus stearothermophilus (Geobacillus stearothermophilus ) spores, Bacillus subtilis ( Bacillus subtilis ) spores, Bacillus atrophaeus ( Bacillus atrophaeus ) spores , Bacillus megaterium spores, Bacillus coagulans spores, Clostridium sporogenes spores, Bacillus pumilus spores, and combinations thereof.

SCBI 100 also includes an ampoule 112 having a first end 114 , a second end 116 , and a side wall 118 . Sidewall 118 is substantially cylindrical and may have an oval or circular cross-section. Ampoule 112 can be made of a fragile or brittle material such as glass or plastic. The first end 114 and the second end 116 are disposed at opposite ends of the side wall 118 and may have the form of a semi-ellipsoid or a hemisphere. Accordingly, first end 114 may be referred to as first dome 114 and second end 116 may be referred to as second dome 116 . Ampoule 112 contains liquid growth medium. The growth medium should be capable of promoting the growth of any living microorganisms or other sources of biological activity disposed on the carrier 110 . Preferably, the microorganism is selected to produce an enzyme that can interact with the enzyme substrate to produce a detectable product, e.g., by having a fluorescence intensity or spectrum that differs from that of other materials in SCBI 100 . Continued growth of the microorganism in the growth medium results in an increase in the concentration of the detectable product in the growth medium. In certain embodiments, the detectable product is a fluorophore. Thus, an increase in the concentration of the detectable product results in an increase in fluorescence. That is, a detectable product is detectable via a change in fluorescence.

Enzymes and enzyme substrates that can be used to detect the efficacy of a sterilization cycle are identified in: US Patent No. 5,073,488, entitled "Rapid Method for Determining Efficacy of a Sterilization Cycle and Rapid Read-Out Biological Indicator", December 17, 1991 Announcement on May 23, 1995, the disclosure of which is incorporated herein by reference; U.S. Patent No. 5,418,167, entitled "Rapid Read-Out Biological Indicator", announced on May 23, 1995, the disclosure of which is incorporated herein by reference; U.S. Patent No. 5,223,401, entitled "Rapid Read-Out Sterility Indicator," issued June 29, 1993, the disclosure of which is incorporated herein by reference; and U.S. Patent No. 9,322,046, entitled "Biological Sterilization Indicator" , announced on April 26, 2016, the disclosure of which is incorporated herein by reference.

Suitable enzymes may include hydrolases and/or enzymes derived from spore-forming microorganisms such as Bacillus subtilis. Enzymes from spore-forming microorganisms that are useful in exemplary biological indicators can include beta-D-glucosidase, alpha-D-glucosidase, alkaline phosphatase, acid phosphatase, butyrate esterase, octanoic acid Esterase lipase, myristate lipase, leucine aminopeptidase, valine aminopeptidase, chymotrypsin, phosphohydrolase, α-D-galactosidase, β-D-galactosidase Glycosidase, tyrosine aminopeptidase, phenylalanine aminopeptidase, β-D-glucuronidase, α-L-arabinofuranosidase, N-acetyl-β-glucosaminidase, β-D- Cellobiosidases, alanine aminopeptidases, proline aminopeptidases, fatty acid esterases, and combinations thereof.

In some exemplary methods for determining the efficacy of a sterilization cycle as disclosed herein, an enzyme substrate is converted to a detectable product. For example, an enzyme substrate can be characterized by a first emission spectrum (eg, a first fluorescence emission spectrum) and a detectable product can be characterized by a second emission spectrum (eg, a second fluorescence emission spectrum).

In some exemplary methods for determining the efficacy of a sterilization cycle as disclosed herein, suitable enzyme substrates used may include luciferase substrates. Useful luciferase substrates may be selected from: fluorogenic 4-methylumbelliferyl derivatives (hydrolyzable to 4-methylumbelliferone ("4-Mu")), Derivatives of 7-amido-4-methylcoumarin, diacetylfluorescein derivatives, fluoresceinamine and combinations thereof.

Exemplary 4-methylumbelliferyl derivatives may be selected from: 4-methylumbelliferyl-2-acetamido-4, 6-O-benzylidene-2-deoxy-β-D-glucose Pirananoside, 4-methylumbelliferyl acetate, 4-methylumbelliferyl-N-acetyl-β-D-aminogalactosaminide (galactosaminide), 4-methylumbelliferyl-N -Acetyl-α-D-glucosamine glycoside, 4-methylumbelliferyl-N-acetyl-β-D-glucosamine glycoside, 2'-(4-methylumbelliferyl)-α- D-N-acetylneuraminic acid, 4-methylumbelliferyl α-L-arabinofuranoside, 4-methylumbelliferyl α-L-arabinoside, 4-methylumbelliferyl butyrate, 4 -Methylumbelliferyl β-D-cellobioside, Methylumbelliferyl β-D-N,N'diacetylchitobioside, 4-methylumbelliferyl elaidic acid, 4-methylumbelliferyl Form group β-D-trehaloside, 4-methylumbelliferyl α-L-trehaloside, 4-methylumbelliferyl β-L-trehaloside, 4-methylumbelliferyl α-D-galactoside Glycoside, 4-methylumbelliferyl β-D-galactoside, 4-methylumbelliferyl α-D-glucoside, 4-methylumbelliferyl β-D-glucoside, 4-methylumbelliferyl β-D-glucoside, 4-methylumbelliferyl Form group β-D-glucuronide, 4-methylumbelliferyl p-guanidinobenzoate, 4-methylumbelliferyl heptanoate, 4-methylumbelliferyl α-D-mannoside, 4-Methylumbelliferyl β-D-Mannoside, 4-Methylumbelliferyl Oleate, 4-Methylumbelliferyl Palmitate, 4-Methylumbelliferyl Phosphate, 4-Methylpropionate Umbelliferyl ester, 4-methylumbelliferyl stearate, 4-methylumbelliferyl sulfate, 4-methylumbelliferyl β-D-N,N',N"-triacetyl chitosan Sugar, 4-methylumbelliferyl 2,3,5-tri-o-benzoyl-α-L-arabinoside, cinnamic acid 4-methylumbelliferyl-p-trimethylammonium, 4-Methylumbelliferyl β-D-xyloside, and combinations thereof.

In certain embodiments, the fluorescent reaction in SCBI can be based on the alpha-glucosidase enzyme naturally present in the spore coat of Geobacillus stearothermophilus and which is believed to have An important enzyme for the germination of Bacillus steatosis. α-Glucosidase can be used to hydrolyze the linkage between the glucose and 4-methylumbelliferyl moieties of 4-methylumbelliferyl α-D-glucopyranoside (α-MUG). α-MUG does not fluoresce. However, after hydrolysis and separation of the fractions, the 4-methylumbelliferone (4-MU) product fluoresces. 4-MU fluoresces when excited by an external energy source, such as a light source emitting light with a wavelength between about 360 and 370 nm. When so excited, 4-MU emits light at a wavelength between about 440 and 460 nanometers. In certain embodiments, the light source emits light at a wavelength of about 365 nm, and the 4-Mu emits light at a wavelength of 450 nm. The fluorescence of 4-MU is pH dependent. For example, when excited by light with a wavelength of 365 nm, the intensity of emitted light is highest at pH 10.3. The intensity decreases with pH until about pH 7. Below this pH, the intensity becomes negligible.

SCBI 100 may also include plug-in 120 . Insert 120 may include a platform 122 having a top surface 124 and a bottom surface 126 . The insert 120 also includes a side wall 127 . The sidewall 127 of the platform 122 may rest on a support surface 128 which may be integrally formed as part of the housing 102 . Sidewall 127 and top surface 124 of platform 122 together define a well 130 configured to receive second end 116 of ampoule 112 . Platform 122 defines therethrough an aperture 150 through which liquid growth medium may travel upon rupture of the ampoule.

SCBI 100 can be assembled according to the following steps. First, a housing 102 is provided. Second, the carrier 110 is placed in the housing 102 such that the carrier rests on the bottom wall 144 of the housing 102 . Third, the insert 120 is placed in the housing 102 so that the sidewall 127 of the platform 122 rests on the support body surface 128 . Alternatively (not shown), in some configurations lacking a support surface 128, the insert 120 may be seated directly on the bottom wall 142 and may at least partially contact the carrier 110. Fourth, the ampoule 112 is inserted into the housing 102 so that the second end 116 contacts the insert 120 . Finally, cap 104 is coupled to housing 102 and ampoule 112 . The protrusion 106 has approximately the same diameter as the ampoule 112 so that a friction fit is formed between the ampoule 112 and the protrusion 106 . So assembled, the central longitudinal axes of the ampoule 112, housing 102, cover 104, and insert 120 are coaxial or substantially coaxial. Other assembly procedures can be performed to achieve the same SCBI 100 configuration.

After the sterilization process, the SCBI 100 can be started and monitored to determine if the sterilization cycle was effective. A compressive force 146 is applied between the housing 102 and the cover 104 to activate the SCBI 100 . Ampoule 112 resists this compressive force because ampoule 112 is in contact with insert 120 , and insert 120 is in contact with support 128 such as housing 102 . When the compressive force applied to the cap 104 is greater than the bursting force that the ampoule 112 can withstand, the ampoule 112 will rupture. Upon rupture of the ampoule 112 , the cover 104 moves to its second position and releases the growth medium to submerge the carrier 110 .

Various features can be included in the SCBI to facilitate activation of the SCBI, for example by reducing the force the user must exert in order to break the ampoule. Exemplary features related to this functionality are disclosed in co-pending US Patent Application Nos. 15/057,768 and 15/397,018.

The activation of SCBI 100 should be confirmed. For example, actuation may be confirmed by checking, for example, that the ampoule 112 is broken, that the growth medium is submerged in the carrier 110, that a substantial volume of growth medium is disposed between the bottom surface 126 of the insert 120 and the bottom wall 144 of the housing 102, and/or Cover 104 is tied in the second position. To increase the likelihood that a boot failure or incorrect boot can be detected, multiple checks can be performed. For example, in addition to checking whether the ampoule 112 is broken, checking whether the growth medium is immersed in the carrier 110 can also be performed. Such checks may be performed by a user or an electromechanical device capable of authenticating SCBI 100 , such as Biological Indicator Analyzer (“BIA”) 200 . To increase the likelihood that a failed or incorrect startup can be detected, various checks should be performed by both the user and the BIA 200 .

FIG. 3 depicts, in block form, an exemplary BIA 200 operable to analyze a biological indicator (eg, SCBI 100 ) that has been subjected to a sterilization cycle. The BIA 200 is configured to test SCBI, collect information about SCBI (e.g., location of growth medium, color of growth medium, light intensity of growth medium), process information, and determine whether a sterilization cycle is effective. BIA 200 includes a plurality of wells 210, each well configured to receive a respective SCBI 100 sample therein. Although two wells 210 are shown, it is understood that any other suitable number of wells may be provided, including eight wells, less than eight wells, or more than eight wells. Each well 210 further includes a heating element 212 that can be used to incubate the SCBI 100 while it is inserted therein. This cultivation promotes the growth of any living microorganisms within the SCBI. In various embodiments, the heating element can bring the temperature of the well to between about 50°C and about 60°C. In some embodiments, the heating element can reach a temperature of about 57°C and cause the SCBI 100 to reach a substantially similar or identical temperature. The BIA 200 also includes a processor 220 operable to execute instructions and control algorithms, process information, and the like.

Each well 210 has an associated light source 230 and sensor 240 . Each light source 230 is configured to project light through the housing 102 of the SCBI 100 inserted into the corresponding well 210 . Each sensor 240 is operable to detect fluorescent light emitted by the growth medium. Each sensor 240 is positioned adjacent to each well 210 such that when the SCBI 100 is disposed in the well, the sensor 240 is adjacent to the bottom surface 126 of the insert 120 and the bottom wall 144 of the housing 102 of the SCBI 100 the part between.

Light source 230 may be in the form of, for example, a laser configured to emit ultraviolet light. In some embodiments, the light emitted by light source 230 has a wavelength of 370 nanometers. Various other suitable forms that light source 230 may take will be apparent to those of ordinary skill in the art in view of the teachings herein. For further example, the sensor 240 may include a charge-coupled device (CCD). Furthermore, it may be a sensor optimized to detect light generated by fluorescence, ie a fluorescent sensor. In some embodiments, the sensor 240 is a silicon photodiode, such as the silicon photodiode S2386-5K manufactured by Hamamatsu. The fluorescence of the growth medium is mainly dependent on the number of living microorganisms contained in the growth medium. Accordingly, sensor 240 is configured to detect the presence of live microorganisms in the growth medium based on the extent to which the growth medium fluoresces in response to light from light source 230 . However, the fluorescence of the growth medium also depends on whether any fluorescence quenching occurs. Fluorescence quenching can also be used to confirm correct activation of SCBI 100, as will be explained in detail below.

BIA 200 optionally further includes user feedback and/or input devices, such as touch screen display 250 . Touch screen display 250 is operable to provide various user interface displays associated with the operation of biological indicator analyzer 200. The touch screen display 250 is further configured to receive user input in the form of a user touching the touch screen display 250 according to conventional touch screen technology. Additionally or alternatively, biological indicator analyzer 200 may include various other types of user input features including, but not limited to, buttons, keypads, keyboards, mice, trackballs, and the like. The display provided through touch screen display 250 may be driven by processor 220 . User input received through touch screen display 250 may be processed by processor 220 .

The BIA 200 of the present example further includes a memory 280, such as a non-transitory storage medium (eg, a hard drive or a flash memory drive), operable to store control logic and instructions for execution by the processor 220 , to drive components such as the light source 230 and the touch screen display 250 , and to perform calculations and analysis of data, especially the data collected by the sensor 240 . Memory 280 may also be used to store user input, data collected by sensors 240, and calculations based on this data.

Fluorescence data collected by the BIA 200 can be used to determine changes in fluorescence over time. This data can be used to determine fluorescence quenching. Fluorescence quenching is a term generally used to describe various processes that cause a decrease in the fluorescence intensity of a substance. For fluorescent substances, such processes include, but are not limited to: 1) heating, 2) lowering the pH; and 3) adding another substance or material known to cause a decrease in fluorescence intensity, sometimes called a "quencher". )". Exemplary quenchers include, but are not limited to, oxygen, aniline, bromobenzene, acrylamide, hydrogen peroxide, imidazole, indole, and succinimide. Quenchers can also be metal ions such as Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Hg ²⁺ , Pb ²⁺ , Ag ⁺ , Cr ³⁺ , and Fe ³⁺ .

When the SCBI 100 is inserted into the well 210 of the BIA 200, its temperature may be substantially equal to the ambient temperature, such as room temperature. However, SCBI 100 may have a warmer than ambient temperature because the vacuum chamber in a sterilization system is often warmer than the ambient temperature at the end of the sterilization cycle. Regardless of the temperature of the SCBI 100, when it is inserted into the well 210 of the BIA 200 or shortly thereafter (eg, about 1 second, 5 seconds, or 15 seconds), the BIA 200 activates the heating element 212 to raise the temperature in the well to about Between 50°C and about 60°C, such that the SCBI 100 reaches a substantially similar or identical temperature. Heating SCBI 100 in this manner quenches the fluorescence of SCBI 100 components. While quenching is most pronounced in the growth medium, it may also be present, albeit to a lesser extent, in other features of SCBI 100, namely non-liquid components including housing 102.

Various components of the SCBI 100 can be tested to determine if the SCBI 100 is properly activated. In a properly activated SCBI 100 , the growth medium should submerge the carrier 110 and be disposed on the bottom of the SCBI 100 between the bottom surface 126 of the insert 120 and the bottom wall 144 of the housing 102 . Accordingly, BIA 200 can assay this portion of SCBI 100 to determine if growth media is present there. For example, BIA 200 may activate light source 230 . In some embodiments, the light emitted by light source 230 has a wavelength of about 370 nanometers. If the growth medium is present, the growth medium will be excited, and the sensor 240 will record the corresponding fluorescence intensity, and output the corresponding voltage to the processor 220 for storage in the memory 280 . However, if the growth medium is not present, the growth medium will not be activated. Nevertheless, the light source may excite other features and materials of the SCBI 100 such that the sensor 240 will record a corresponding intensity and output a corresponding voltage to the processor 220 for storage in the memory 280 . The intensity recorded by the sensor 240 depends on whether the growth medium is present at the bottom of the properly primed SCBI 100, or on the absence of the growth medium at the bottom of the incorrectly primed SCBI 100 (so that the growth medium remains in, for example, an unruptured In the ampoule 112, it will be different outside the assay zone where the light source 230 and sensor 240 can interrogate).

Processor 220 may be programmed to detect the presence of growth medium at the bottom of SCBI 100 . In some embodiments, threshold values corresponding to light and/or fluorescent intensity may be stored in memory 280 . Specifically, the light and/or fluorescent intensity corresponding to the liquid in the bottom of SCBI 100 (between bottom surface 126 of insert 120 and bottom wall 144 of housing 102 ) may be stored in memory 280 . By comparing the measured intensity with a threshold value, it can be determined whether the SCBI 100 is properly activated. For example, processor 220 may be programmed to determine whether the measured intensity falls between a minimum threshold and a maximum threshold. An intensity measurement that falls between the thresholds will indicate that the growth medium is set at the bottom of the SCBI 100 and that the SCBI 100 has started up correctly. An intensity measurement that falls below the minimum threshold would indicate that the growth medium was not disposed at the bottom of the SCBI 100, possibly because it remained in the unruptured ampoule 112 due to improper priming. Intensity measurements that fall above the maximum threshold may indicate a malfunction within the BIA 200 . In an embodiment where light source 230 provides light at a wavelength of 370 nm, the minimum threshold may be about 0.02 μW/cm ² and the maximum threshold may be 0.10 μW/cm ² . In an embodiment where sensor 240 is a Hamamatsu silicon photodiode S2386-5K, these minimum and maximum values should be output from sensor 240 at 0.47 volts and 2.2 volts, respectively. In some embodiments, the intensity measurements used to confirm proper activation are taken immediately, or up to about 300 seconds after insertion of the SCBI 100 into the well 210 . In certain embodiments, the intensity measurement is taken about 70 seconds after the SCBI 100 is inserted into the well 210 . SCBI activation can be determined in this manner with an accuracy of at most about 90% to 95%. Variations in intensity from light source 230 and the temperature in SCBI 100 maintained by heating element 212 may prevent greater accuracy from being achieved.

Therefore, it is suggested to supplement this form of confirmation with other methods and forms of confirmation, such as visual confirmation performed by the user, or the following methods based on quenching effects of the growth medium and other components in the SCBI 100 .

Quenching can be determined by calculating the difference or ratio between a first fluorescence intensity measurement taken at a first time and a second fluorescence intensity measurement taken at a second time. Quenching effects are generally more pronounced in liquids than in solids because molecules in liquids collide more frequently than molecules in solids. Plastic materials (especially clear or transparent plastic materials) commonly used in medical devices, including polycarbonate and cycloolefin, exhibit relatively small decrease in fluorescence due to heating compared with colored growth media, including Those containing 4-MU, such as those used in SCBI 100. For example, after being heated from room temperature to between 50°C and 60°C and maintained at a higher temperature for about four minutes, the quenching effect (that is, the decrease in fluorescence intensity) exhibited by the plastic material is between about 0 and 5%. , while the growth medium exhibits a quenching effect between about 5% and 25%.

Thus, BIA 200 can be used to: 1) take a first fluorescence intensity measurement at a first time; 2) quench the fluorescence of SCBI 100 by heating SCBI 100; 3) take at a second time after the first time second fluorescence intensity measurement result; 4) by calculating the difference or ratio between the first fluorescence intensity measurement result and the second fluorescence intensity measurement result, comparing the two measurement results to determine the degree of quenching; 5) determination Whether the degree of quenching corresponds to quenching from only solid or non-liquid components of the SCBI 100, or additionally to quenching from the growth medium; and 6) an indication of whether the SCBI 100 was activated incorrectly.

The first fluorescence intensity measurement may be taken at a first time (ie, between about 0 seconds and about 100 seconds after insertion of the SCBI 100 into the well 210). The second fluorescence intensity measurement can be taken at a second time (ie, between about 0 seconds and about 300 seconds after the first time). In certain embodiments, the first time is about 70 seconds after the SCBI 100 is inserted into the well 210, and the second time is about 210 seconds after the first time (or 280 seconds after the SCBI 100 is inserted into the well 210).

The comparing and determining steps can be performed by the processor 220 in various ways. For example, processor 220 may calculate the difference between the second measurement and the first measurement and compare the difference to threshold values stored in memory 280 corresponding to the degree of quenching from the solid material and growth medium. Alternatively, processor 220 may calculate a ratio between the second measurement and the first measurement and compare the ratio to threshold values stored in memory 280 corresponding to the degree of quenching from the solid material and growth medium. When the BIA 200 performs two start-up checks (one to determine whether a single intensity value is between the expected minimum and maximum threshold values, and the other to determine whether the two intensity values correspond to the expected degree of fluorescence quenching), Proper startup can be confirmed with a high degree of accuracy, at least up to 99%.

The step of indicating may take the form of processor 220 causing a message to be displayed on display 250 indicating whether SCBI 100 was activated correctly or incorrectly. Alternatively or in addition, BIA 200 may sound an alarm upon determining that SCBI 100 has been activated improperly.

To enhance the difference in the degree of quenching experienced by the non-liquid component and the growth medium when subjected to heating, the quenching properties of the non-liquid component and the growth medium can be modified. Specifically, the effect of heating on quenching can be increased for growth media and decreased for non-liquid components. This facilitates the differentiation of non-liquid components from the growth medium, based on quenching calculations from light intensity measurements taken by sensor 240 . In turn, such modifications can increase the reliability of determining whether the SCBI 100 has properly activated based on quench calculations. For example, quenchers such as aniline, bromobenzene, acrylamide, hydrogen peroxide, imidazole, indole, or succinimide can be added to the growth medium, eg, by admixture. Oxygen can also be considered a quencher. The oxygen content of the growth medium at sea level was about 37 mg/L. Accordingly, the oxygen content can be increased to about 40 mg/L, about 45 mg/L, or higher to increase quenching of the growth medium by heat. Metal ions can also be considered as quenchers. Exemplary metal ion quenchers include: Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Hg ²⁺ , Pb ²⁺ , Ag ⁺ , Cr ³⁺ , and Fe ³⁺ .

Additionally or alternatively, the growth medium can be modified to have a higher pH. In certain embodiments, the pH of the growth medium is between about 7.7 and about 8.7. In certain embodiments, the pH of the growth medium can be about 8.2. However, the quenching effect is generally maximized at a pH of about 10 because at this pH the fluorescence intensity is also maximized. Thus, the pH of the growth medium can be increased to about 8.5, about 9, about 9.5, and about 10.

In order to reduce the amount of quenching caused by heat in the non-liquid components of SCBI 100 (e.g., housing 102 and insert 120), the non-liquid components can be made of materials with low concentrations of antioxidants (such as cyclic olefins) and present in the presence of possible absorption from Any other UV-absorbing compound in the components of the UV light for which BIA 200 performs the verification. Non-liquid components can also be made of UV transparent materials such as quartz or low density polyethylene.

Larger increases in temperature can also be utilized to assist in differentiating non-liquid components from growth media based on quenching measurements. Since the degree of quenching varies with temperature, the quenching caused by large temperature changes is generally greater than that caused by small temperature changes. Although this change affects the quenching of both the growth medium and the non-liquid components of SCBI 100, the effect of the temperature change on the quenching of the growth medium is greater than that of the non-liquid components. Thus, by maximizing the temperature difference subject to the design constraints associated with assessing microbial growth, higher accuracy can be achieved in distinguishing quenches corresponding to non-liquid components from the growth medium. Thus, BIA 200 can heat SCBI 100 to temperatures above 60°C to impart a greater quenching effect on SCBI 100, further accentuating any difference in fluorescence intensity imparted by heating. In a similar situation, the BIA 200 may include cooling elements beside the well 210, which may be used to cool the SCBI 100 prior to heating, to increase the resulting change in temperature, thereby increasing the concomitant quenching effect.

Quenching in the growth medium can be counteracted by an increase in fluorescence resulting from a fluorescent product produced by the enzyme in the growth medium. Thus, the amount of enzyme produced within the growth medium should be minimized, subject to the design constraints required to determine the growth of the microorganism associated with the enzyme produced.

FIG. 4 depicts a flowchart illustrating an exemplary method 300 for determining whether a biological indicator, such as SCBI 100 , has been properly activated in accordance with some of the foregoing teachings. This method can be performed as part of a larger method (eg, as a subroutine) in which the BIA 200 monitors SCBI for changes in fluorescence caused by microbial growth. While each and every teaching above is not expressly incorporated into this example method, it should be understood that teachings not expressly stated can be incorporated into a method for determining activation of a biological indicator such as SCBI 100 based on a quenching effect . Also, while the method is presented with reference to the SCBI 100, BIA 200, and components thereof, it should be understood that the method can be practiced with other biological indicators than the SCBI 100 and other biological indicator analyzers than the BIA 200 .

Method 300 begins at step 310 , where a healthcare professional applies compressive force 146 between housing 102 and cover 104 to activate SCBI 100 . In typical usage, step 310 occurs after subjecting the SCBI to a sterilization procedure. Successful actuation causes cover 104 to move from the first position to the second position, thereby breaking ampoule 112 and allowing the growth medium contained therein to flow to the bottom of SCBI 100, between bottom surface 126 of insert 120 and bottom wall 144 of housing 102. between. In step 320 , the healthcare provider inserts the SCBI 100 into the well 210 of the BIA 200 . In step 330 , BIA 200 activates heating element 212 . In step 340 , BIA 200 performs a first verification of the bottom of SCBI 100 by exciting the SCBI with light source 230 and measuring a first light intensity with sensor 240 . Step 340 may be performed between about 0 seconds and about 100 seconds after step 320 . In some embodiments, step 340 may occur before step 330 . In other embodiments, step 340 may occur after step 330 . In some embodiments, step 340 may be performed about 70 seconds after step 320 , while in other embodiments, step 340 may be performed about 70 seconds after step 330 . In step 350 , the measured first light intensity output by the sensor 240 at the first voltage value is stored in the storage device 280 . In step 360 , the processor 220 compares the first voltage value (V ₁ ) with the minimum threshold voltage value (V _min ) and the maximum threshold voltage value (V _max ). In an embodiment where light source 230 emits light at a wavelength of 370 nm and sensor 240 is a silicon photodiode S2386-5K manufactured by Hamamatsu, the minimum threshold voltage value may be between about 0.4 and about 0.5 volts. For example, the minimum threshold voltage value may be about 0.47 volts. The maximum threshold voltage value may be between about 2.1 and about 2.3 volts. For example, the maximum threshold voltage value may be about 2.2 volts. If the first voltage value is less than the minimum threshold voltage value or greater than the maximum threshold voltage value, the SCBI 100 may have improperly started, or the BIA 200 may have malfunctioned. Therefore, in step 370, the method is aborted. An error message may be displayed on the display 250 or an alarm may sound. If the first voltage value falls between the minimum threshold and the maximum threshold, the SCBI 100 may have started correctly. To confirm that the SCBI 100 was properly started, the BIA 200 continues the method.

The BIA 200 performs a second verification of the SCBI 100 in step 380 . For step 380 , BIA 200 excites the SCBI using light source 230 and measures a second light intensity using sensor 240 . Step 380 may be performed between about 0 seconds and about 300 seconds after step 340 . For example, step 380 is performed about 210 seconds after step 340 (ie, about 280 seconds after turning on the heating element 212). In step 390 , the measured second light intensity output by the sensor 240 at a second voltage value (V ₂ ) is stored in the storage device 280 .

In step 400, processor 220 calculates a quenching metric. For example, the quenching metric may be a "quenching difference", ie the difference between the second voltage value and the first voltage value, or it may take the form of a "quenching ratio", also That is, the ratio between the second voltage value and the first voltage value. If the fluorescence of the SCBI 100 has not been quenched, the second voltage value should be equal to or approximately equal to the first voltage value. Accordingly, the quench difference ("QD") should be equal to or approximately equal to zero, and the quench ratio ("QR") should be equal to or approximately equal to one. As shown in step 400, the measure of quenching is the quenching ratio. In step 410, the processor determines whether there has been minimal quenching (e.g., a quenching ratio greater than about 95%) or significant quenching (e.g., a quenching ratio between 75% and 95% when SCBI 100 is heated to about 57°C %, the first voltage value corresponds to a first light intensity measured about 70 seconds after activating the heating element 212, and the second voltage value corresponds to a second light intensity measured about 280 seconds after activating the heating element 212). Minimal quenching indicates that the SCBI 100 was not properly primed, as the growth medium should undergo quenching when subjected to heat. When the processor 220 calculates the minimum quench, step 420 is performed, where the method is aborted. An error message may be displayed on the display 250 or an alarm may sound. However, when processor 220 calculates significant quenching, BIA 200 begins evaluating SCBI 100 according to its primary purpose, namely monitoring for further changes in fluorescence of the growth medium that may be attributable to microbial growth, as shown in step 430 .

It should be appreciated that any of the examples and/or embodiments described herein may include various other features in addition to or instead of those described above. The teachings, representations, embodiments, examples, etc. described herein should not be considered in isolation with respect to each other. Various suitable ways in which the teachings herein may be combined should be apparent to those of ordinary skill in the art in view of the teachings herein.

Having shown and described exemplary embodiments of the subject matter contained herein, further adaptations of the methods and systems described herein may be made by appropriate modifications without departing from the scope of the patent scope of this application. Some such modifications should be apparent to those of ordinary skill in the art. For example, the examples, embodiments, geometries, materials, dimensions, ratios, steps, and the like discussed above are illustrative. Accordingly, claims should not be limited to the specific details of structure and operation illustrated in the written description and drawings.

100‧‧‧Self-contained biological indicator

102‧‧‧Shell

104‧‧‧Cover

106‧‧‧convex part

110‧‧‧carrier

112‧‧‧ampoules

114‧‧‧first end/first dome

116‧‧‧Second End/Second Dome

118‧‧‧side wall

120‧‧‧plug-ins

122‧‧‧platform

124‧‧‧top surface

126‧‧‧bottom surface

127‧‧‧side wall

128‧‧‧support surface/support

130‧‧‧well

144‧‧‧bottom wall

150‧‧‧holes

Claims (8)

A method for analyzing a biological indicator comprising a non-liquid component and an ampoule containing a liquid growth medium, the method comprising: breaking the ampoule; positioning the biological indicator to a biological indicator analyzer In, the biological indicator analyzer has a heating element, a light source and a fluorescent sensor; activate the heating element; measure a first fluorescence intensity of the biological indicator; quenching the intensity from the first fluorescence intensity to a second fluorescence intensity; measuring the second fluorescence intensity; calculating a quenching metric from the second fluorescence intensity and the first fluorescence intensity; comparing the quenching metric and a threshold value; based on comparing the quenching metric to the threshold value, determining that the quenching metric indicates quenching from the non-liquid component rather than the liquid growth medium; and in response to determining that the quenching metric is indicative of quenching from the non-liquid Quenching of components other than the liquid growth medium discontinues further monitoring of fluorescence by the biological indicator analyzer; wherein the quenching measure comprises a quenching ratio or a quenching difference; wherein the quenching ratio comprises the first a ratio of fluorescence intensity to the second fluorescence intensity, and determining that the quenching measure indicates quenching from the non-liquid component rather than the liquid growth medium comprises determining that the quenching ratio is greater than about 95%; and wherein the The quenching difference comprises the difference between the second fluorescence intensity and the first fluorescence intensity, and the step of determining that the quenching measure indicates quenching from the non-liquid component rather than the liquid growth medium comprises comparing the quenching difference and thresholds corresponding to the degree of quenching of the non-liquid component and the liquid growth medium. The method according to claim 1, wherein the step of quenching the fluorescence intensity of the biological indicator comprises heating the biological indicator. The method as claimed in claim 1, wherein the step of quenching the fluorescent intensity of the biological indicator comprises changing the growth medium and the shell of the biological indicator from about 22 degrees Celsius to Heating from a temperature between 25 degrees Celsius to a temperature between about 50 degrees Celsius and 60 degrees Celsius. The method of claim 2, further comprising the step of cooling the biological indicator prior to the step of positioning the biological indicator in the biological indicator analyzer. The method according to claim 2, wherein the first fluorescence intensity is measured after the heating element is activated, and the second fluorescence intensity is measured after the first fluorescence intensity is measured. The method as claimed in claim 5, further comprising the step of confirming that the first fluorescence intensity value is between a minimum threshold value and a maximum threshold value. The method of claim 1, wherein the first fluorescence intensity is measured at a time between about 0 seconds and about 100 seconds after the biological indicator is positioned to the biological indicator analyzer, and after the first time The second fluorescent intensity is measured at a time between about 0 seconds and about 300 seconds. The method of claim 7, wherein the first time is about 70 seconds after the biological indicator is located in the biological indicator analyzer, and the second time is about 210 seconds after the first time.

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